大豆異黃酮genistein及daidzein對於高血尿酸小鼠模式之腎臟保護作用

The protective effects of soy isoflavones genistein and daidzein against nephropathy in a mouse model of hyperuricemia.

林巧怡(Chiao-Yi Lin)

指導教授 ： 劉承慈

中山醫學大學/健康管理學院/營養學系/碩士(2020年)

https://doi.org/10.6834/csmu202000236

摘要

近年研究顯示，增加膳食大豆攝取量與降低高血尿酸及其共病症之發生風險有關，如：高血壓、代謝症候群、糖尿病。雖然已知大豆異黃酮─genistein及daidzein的多重生物學活性，但尚不清楚其是否對於尿酸誘發之腎損傷具有保護效果。本研究目的是以給予高尿酸飲食之小鼠模式，觀察介入genistein或daidzein對於小鼠腎臟發炎反應、腎臟尿酸轉運蛋白表現、腎功能指標及腎臟組織形態學之影響。本研究於十週齡雄性C57BL/6小鼠，以含2% potassium oxonate（w/w）及3% uric acid（w/w）之soy-free diet餵食小鼠十天提升血尿酸值，並將其隨機分為7組，自第一天起分別每日灌食載劑丙二醇（0.05 ml/隻，HUA組）、高或低劑量genistein（160、10 mg/kgw，分別為GH、GL組）、高或低劑量daidzein（160、10 mg/kgw，分別為DH、DL組）、allopurinol（5 mg/kgw，allopurinol組），並與餵食soy-free diet且灌食丙二醇之小鼠（ND組）對照。於灌食第十天當晚禁食12小時後，各誘發組以腹腔注射 potassium oxonate（300 mg/kgw）及胃灌 hypoxanthine（500 mg/kgw）以進一步提升小鼠血尿酸濃度，ND組則處以載劑，並在2.5小時之後以二氧化碳犧牲，同時收集血液及腎臟組織進行分析。結果發現給予高尿酸飲食，小鼠之血清尿酸濃度僅微幅上升，但可顯著降低腎臟中nitrite/nitrate含量，而高劑量之daidzein可顯著降低血清中尿酸濃度且高和低劑量之daidzein均可顯著回升腎臟中nitrite/nitrate含量。在腎臟發炎反應方面，Western blotting分析結果顯示，高尿酸飲食造成小鼠腎臟中TLR4、MD2及MyD88蛋白質表現量均顯著上升，介入兩種大豆異黃酮不論高或低劑量均可顯著逆轉高尿酸飲食誘發之上述蛋白質表現。在腎臟尿酸轉運蛋白表現方面，根據IHC分析腎臟組織結果顯示，給予小鼠高尿酸飲食會使腎臟尿酸再吸收轉運蛋白─URAT1蛋白質表現量顯著上升及分泌轉運蛋白─OAT1蛋白質表現量顯著下降，而介入genistein及daidzein後，可降低URAT1蛋白質表現量及提升OAT1蛋白質表現量。在腎功能指標方面，給予高尿酸飲食顯著造成小鼠血清creatinine濃度上升，介入高劑量之genistein及daidzein皆可顯著降低血清中creatinine濃度。在腎臟組織形態學方面，由H E染色發現，本研究模式下腎臟之腎絲球及腎小管皆未因高尿酸飲食或介入飲食造成其組織形態之改變；但由PAS染色發現，給予高尿酸飲食之小鼠腎臟腎絲球PAS染色程度增加，介入大豆異黃酮皆可使PAS之染色結果呈現緩和的趨勢。綜合以上結果可知，genistein及daidzein兩種大豆異黃酮均能逆轉高尿酸飲食誘發的腎臟促發炎訊號分子表現並可能透過調節腎臟尿酸轉運蛋白表現促進尿酸排出，且daidzein尚可能逆轉高尿酸飲食誘發之腎臟血壓調控異常，因此genistein和daidzein具有應用於尿酸相關腎損傷保護作用之潛力。

關鍵字

氧嗪酸鉀 ； 大豆異黃酮 ； 金雀異黃酮 ； 大豆異黃酮苷素 ； 腎損傷

並列摘要

Recent studies have shown that increased dietary soybean intake is associated with lowered risk of hyperuricemia and its comorbidities, such as hypertension, metabolic syndrome, and diabetes. Although the multiple biological activities of soy isoflavones─genistein and daidzein are known, it is not clear whether they have protective effects on uric acid-induced nephropathy. The purpose of this study was to investigate the effects of interventional genistein or daidzein on kidney inflammation, renal uric acid transporter expression, renal function indicators and renal tissue morphology in a high-uric acid (HUA) diet mouse model. Ten-week-old male C57BL/6 mice were randomly assigned to 7 groups, one of which was given normal soy-free diet, and the others were fed a soy-free diet containing 2% potassium oxonate (w/w) and 3% uric acid (w/w) for ten days. From the first day on, 6 groups that fed with HUA diet were gavaged with vehicle (propylene glycol 0.05 ml), GH (genistein 160 mg/kgw), GL (genistein 10 mg/kgw), DH (daidzein 160 mg/kgw), DL (daidzein 10 mg/kgw), or allopurinol (5 mg/kgw), respectively. ND Group was fed a normal soy-free diet and gavaged with propylene glycol. After fasting for 12 hours over night, each induction group was given intraperitoneal injection of potassium oxonate (300 mg/kgw) and gavaged hypoxanthine (500 mg/kgw) to further increase the blood uric acid concentration of mice, and ND group treated with vehicle. After 2.5 hours, mice were sacrificed with carbon dioxide. Blood and kidney were collected for analysis. The results showed that although HUA diet-induced elevation of serum uric acid concentration was only slightly, it significantly reduced the renal nitrite/nitrate content. High-dose daidzein significantly reduced the serum uric acid concentration and both high and low-dose of daidzein significantly reversed renal nitrite/nitrate content. In the other hand, western blotting analysis showed that the HUA diet caused a significant increase in the protein expression of TLR4, MD2 and MyD88 which was significantly reversed by both soy isoflavones regardless of high or low doses. In addition, according to the results of IHC analysis, HUA diet significantly increased the expression of URAT1 protein and decreased the expression of OAT1 in kidney. The altered expression of uric acid transporter was reversed by genistein and daidzein. Finally, HUA diet significantly increased the serum creatinine concentration in mice but was significantly reversed by high-dose genistein and daidzein. The result of H E staining showed that renal morphology of glomeruli and tubules remained unchanged by HUA diet. However, PAS staining found that the HUA diet did increase the staining intensity of glomeruli, while both soy isoflavones can alleviate the PAS staining intensity. In summary, both genistein and daidzein can reverse the expression of HUA diet-induced renal pro-inflammatory signal molecules and may promote uric acid excretion by regulating the expression of renal uric acid transporter. Additionally, daidzein may reverse the HUA diet-induced abnormal regulation of renal blood pressure. Consequently, genistein and daidzein have the potential to be used to prevent uric acid-associated nephropathy.

並列關鍵字

potassium oxonate ； soy isoflavone ； genistein ； daidzein ； nephropathy

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